The three functional Ras genes encode highly related proteins (p21.sup.ras), of 188 or 189 amino acids. See generally Watson, Molecular Biology of the Gene, Fourth Edition, p.1065, et seq. After post-translational modification, the Ras proteins become associated with the inner surface of the plasma membrane. The Ras proteins are known to bind guanine nucleotides (GTP and GDP) and to possess GTPase activity. The GTP bound form of the Ras protein is the biologically active form, i.e. the signal transducing form. The hydrolysis of bound GTP to GDP results in the production of the inactive GDP bound form. Normal Ras proteins will remain in their inactive state until they receive a stimulus from an upstream exchange activity, which results in the exchange of GDP for GTP, followed by a change in the conformation of the Ras protein to its active state. The Ras proteins also have an intrinsic GTPase activity. This GTPase activity catalyzes the hydrolysis of GTP, which converts the active Ras protein to its inactive GDP-bound form.
It is believed that the Ras proteins play an essential role in the control of the mammalian cell cycle. In studies utilizing microinjection techniques, monoclonal antibodies against p21.sup.ras cause G1-induced arrested cell growth. Resting 3T3 cells injected with anti-Ras antibodies are unable to enter the S phase when they are stimulated to divide, while dividing 3T3 cells injected with the antibody are able to complete rounds of DNA synthesis in progress but fail to initiate new rounds.
The overexpression of p21.sup.ras has been shown to lead to transformation of certain cell types. In addition, ras genes mutated at certain residues are also transforming. For instance, mutations in the gens leading to changes in the amino acid residues 12 and 61 can cause transformation. Also, mutations in the gens leading to the replacement of glycine with any amino acid except proline results in an activated or transforming gens. Activating mutations of p21 have been shown to result in the formation of increased levels of GTP-p21.sup.ras, the active form.
Mutations in the Ras genes which lead to the formation of actived p21.sup.ras have been found in many tumor types, in particular pancreatic and colonic carcinomas where ras mutations are found in the majority of these tumor types, Bos, J. K. Cancer Research, 49, 4682-4689 (1989). This suggests that activated Ras genes may contribute to the development of tumors.
Neurofibromatosis is a common autosomal dominant disorder in humans. The protein encoded by a gene associated with Neurofibromatosis is termed NF1. The amino acid sequence of NF1 is set forth at SEQ ID NO: 1. It is a protein of 2818 amino acid residues, and has a 338 amino acid domain termed NF338 with 26% sequence identity with the C-terminal domain of a protein referred to as GAP1. The 709-1044 amino acid domain of GAP1 is known in the literature as "GAP1C". GAP, an acronym for "GTPase activating protein", refers to a class of proteins which have the ability to stimulate GTPase activity. Both GAP1 and NF1 have been shown to stimulate the normal Ras protein's GTPase activity. See Trahey, M. et al., Science, 238, 542-545 (1987); Vogel, U. S. et al., Nature 335, 90-39 (1988); Xu, G. et al., Cell 62, 599-609 (1990); Xu, G. et al., Cell 63,835-841 (1990); Martin, G. et al., Cell 63,848-849 (1990); and Marchuk, D. et al., Genomics 11, 931-940 (1991).
The instrinsic GTPase activity of the transforming mutants of p21.sup.ras is not activated by GAP1 or NF1. Therefore, oncogenic Ras mutants, such as V-HaRas, remain in an active form, i.e., the GTP bound form, for much longer periods than does normal Ras. As a result, these mutant ras proteins have more potent transforming activities than normal p21.sup.ras.
There have been various attempts made to attenuate, inhibit, reverse, or otherwise interfere with the effects of ras transformation. In recent experiments, ras transformed NIH/3T3 cells have been tested with the hope of finding a cure and/or therapeutic approach to Ras-associated cancers. These attempts have included screening tumor suppressor genes which have been found to be able to reverse ras induced malignant transformation by DNA-mediated transfection and overexpression, as detailed in the following paragraph.
Several distinct tumor suppressor genes have been reported to reverse malignancy caused by the Ras oncogenes. See Contente, S. et al., Science 249, 796-798 (1990) and Buettner, R. et al., Mol. Cell. Biol. 11, 3573-3583 (1991): the full length human Rap1 (also called Krev1) which encodes a Ras related G protein of 184 amino acids, Kitayama, H. et al., Cell 56, 77-84 (1989), which tightly binds GAP1 but without any stimulation of its intrinsic GTPase activity, Hata Y, et al., J. Biol. Chem, 13, 265, 7104-7107 (1990); a truncated V-Jun encoding only the C-terminal domain of 150 amino acids (residues 147 to 296) which lacks the N-terminal transactivating domain, Lloyd A. et al., Nature 352, 635-638; the full length murine Thy-1 gene encoding a cell surface glycoprotein of 142 amino acids which is covalently linked to a glycophosphatidyl inositol, Sugimoto, Y. et al., Cancer Research, 51, 99-104 (1991); a human c-Ets-2 DNA encoding the C-terminal DNA-binding domain of 133 amino acids (residues 333 to 466) which binds to Ras responsive DNA element (RRE) in enhancers of several Ras-transactivated genes, Langer S. J. et al., Mol. Cell. Biol. 12, 5355-5362 (1992); and a rat B-myc DNA encoding the N-terminal DNA-binding domain of 120 amino acids which acts as a c-Myc antagonist, Resar, L. M. S. et al., Mol. Cell. Biol. 1130-1136 (1993). None of these gene products reverse transformation by binding directly to p21.sup.ras.
All attempts to reverse malignant transformation caused by oncogenic Ras mutants with either the full length GAP1 or its C-terminal GTPase activating domain (GAP1C) have been unsuccessful, although GAP1C (but not full length GAP1) is able to reverse the malignant transformation caused by overexpression of normal p21.sup.ras, Zhang, K. et al., Nature 346, 754-756 (1990). NF338 is also able to activate normal Ras GTPase activity, Wiesmueller, L. et al., J. Biol. Chem. 267, 10207-10210 (1992); Nur-E-Kamal M. S. A., et al., Mol. Biol. Cell, 3, 1437-1442 (1992) and was shown to bind the Ras protein much more tightly than the GAP1C, Martin, G. et al., Cell 63, 843-849 (1990). The NF338 fragment, which is similar but slightly larger than GAP1C, was reported to reduce the V-HaRas induced heat shock susceptibility of yeasts, Ballester, R. et al., Cell 63, 851-859 (1990).
Therefore a molecule that is capable of reversing or interfering with activated Ras induced malignant transformation was sought. It was discovered that a fragment of NF1 had the ability to attentuate, reverse or otherwise, interfere with Ras transformation. The overexpression of an NF1 fragment in V-HaRas transformed cells greatly reduced the cells' ability to form colonies in soft agar. This ability to form soft agar colonies is known to be closely associated with the cell's malignancy.
It was also found that the NF1 fragment's ability to reverse or interfere with activated Ras transformation was not based on the fragment's ability to act as a GTPase activating protein. Specifically, the NF56 fragment was found to be capable of reversing Ras transformation but is not capable of GTPase activation, as described in the discussion, infra.
Thus, it is the principal object of this invention to provide for a mechanism to reverse activated Ras transformation. The mechanism may include utilizing certain NF1 fragments which bind to p21.sup.ras to prevent interaction between the Ras protein and its downstream target.
Specifically, certain fragments of NF1, i.e., NF1 polypeptides and peptides, possess the ability to reverse Ras induced malignant transformation. The invention includes NF1 fragments and the isolated nucleic acid molecules which code for the fragments.
It is also an object of this invention to provide for expression vectors containing the nucleic acid molecules which code for such fragments of NF1.
It is a further object of this invention to provide for cells which are transfected with expression vectors containing nucleic acid molecules which code for fragments of NF1.
It is yet another object of this invention to provide for a protein complex containing the relevant NF1 fragments and all or part of a Ras protein.
Other objects of this invention include a method for reversing Ras induced transformation and a method for treating Ras induced conditions with pharmaceutical compositions containing the NF1 fragments, in addition to methods for determining whether other molecules are capable of reversing activated Ras induced transformation.